Comprehensive Phytohormone Profiling of Kohlrabi during In Vitro Growth and Regeneration: The Interplay with Cytokinin and Sucrose

Abstract

The establishment of an efficient protocol for in vitro growth and regeneration of kohlrabi (Brassica oleracea var. gongylodes) allowed us to closely examine the phytohormone profiles of kohlrabi seedlings at four growth stages (T1–T4), additionally including the effects of cytokinins (CKs)—trans-zeatin (transZ) and thidiazuron (TDZ)—and high sucrose concentrations (6% and 9%). Resulting phytohormone profiles showed complex time-course patterns. At the T2 stage of control kohlrabi plantlets (with two emerged true leaves), levels of endogenous CK free bases and gibberellin GA20 increased, while increases in jasmonic acid (JA), JA-isoleucine (JA-Ile), indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) peaked later, at T3. At the same time, the content of most of the analyzed IAA metabolites decreased. Supplementing growth media with CK induced de novo formation of shoots, while both CK and sucrose treatments caused important changes in most of the phytohormone groups at each developmental stage, compared to control. Principal component analysis (PCA) showed that sucrose treatment, especially at 9%, had a stronger effect on the content of endogenous hormones than CK treatments. Correlation analysis showed that the dynamic balance between the levels of certain bioactive phytohormone forms and some of their metabolites could be lost or reversed at particular growth stages and under certain CK or sucrose treatments, with correlation values changing between strongly positive and strongly negative. Our results indicate that the kohlrabi phytohormonome is a highly dynamic system that changes greatly along the developmental time scale and also during de novo shoot formation, depending on exogenous factors such as the presence of growth regulators and different sucrose concentrations in the growth media, and that it interacts intensively with these factors to facilitate certain responses.